The present invention relates to assays which use electrochemiluminescence for the detection of helicase activity. Therefore, the invention provides a helicase assay that is rapid, sensitive and suitable for high throughput screening for helicase activity in general, and especially for potential helicase inhibitors. Helicase inhibitors represent a class of pharmacological agents useful for the treatment of disease.
DNA Helicases
DNA helicases are enzymes that are involved in all aspects of nucleic acid metabolism (Lohman and Bjornson, Annu. Rev. Biochem., 1996, 65, 169-214; Matson, S. W., Progress in Nuclear Acid Research and Molecular Biology, 1991, 40, 289-326). The stable form of most DNA in vivo is double stranded helical DNA. Single stranded DNAs are required for DNA replication, repair, recombination and conjugation. In order to form single stranded DNA from a double strand, the DNA duplex must be at least partially unwound and separated. Unwinding of stable duplex DNAs is catalyzed by DNA helicases using the energy derived from the hydrolysis of nucleoside 5xe2x80x2-triphosphates (NTP). Helicase enzymes translocate along DNA to unwind (unhybridize) the duplex stands at a rate that can be as fast as 500-1000 bp/s.
DNA helicases have been identified in various prokaryotes, eukaryotes, bacteriophages and viruses. Most organisms encode multiple helicases. For example, E.coli encodes at least 12 different helicases (Matson, et al., BioEssays, 1994, 16, 13-22) and S. cerevisiae encodes at least six helicase forms (Li, et al., Chromosoma, 1992, 102, S93-S99). Although these DNA helicases play many different functions in DNA metabolism, it is now clear that helicase activity is the function ascribed to unwinding the DNA duplex. Due to the essential function of helicases for cellular metabolism, these enzymes represent important targets for developing therapeutic agents.
Helicase Assays
Several types of assays have been developed to measure the unwinding of duplex nucleic acids by helicases. One such assay measures the sensitization of labeled duplex DNA to single-strand specific nucleases, such as S1 or exonuclease I, resulting in the production of ssDNA during unwinding (Palas et al., J. Biol. Chem., 1990 265:3447). Electron microscopy has also been employed to visualize directly regions of DNA unwound by proteins such as recBCD enzyme, rep protein, E. coli helicases I and II, and SV40 T antigen (Runyon et al., Proc. Natl. Acad. Sci., 1990, 87:6383).
The most common assay for determining helicase activity in vitro utilizes electrophoresis (Venkatesanet al., J. Biol. Chem., 1982, 257, 12426; Matson, et al., J. Biol. Chem., 1983, 258, 14017). Helicase enzyme unwinds duplex DNA yielding single stranded DNAs. The products of this reaction are resolved on a polyacrylamide or agarose gel. A radioactive label within the DNA permits direct visualization and quantitation of the results.
A continuous spectrophotometric assay developed for studying recBCD enzyme helicase activity utilizes a ssDNA binding protein, either E. coli SSB protein (single-strand DNA binding protein) or phage T4 gene 32 protein, as the reporter molecule (Roman et al., Biochemistry, 1989, 28:2863). As dsDNA is unwound, the SSB protein binds to the ssDNA formed, resulting in quenching of its intrinsic fluorescence. Additional helicase assays are described in U.S. Pat. Nos. 4,568,649, 5,705,344 and 5,747,247.
Each of these assays described above have certain limitations relating to sensitivity, number of steps required to perform the assay, and/or the limited number of samples that can be tested. Obviously, such methods are not readily adaptable for highly sensitive, high-throughput drug screening formats. Therefore, there is a need in the art for a simple, yet sensitive helicase assay that can be performed in high throughput. The present invention overcomes these obstacles, and provides a helicase assay that is rapid, sensitive and suitable for a high throughput screening for potential helicase inhibitors.
Electrochemiluminescence Assays
Electrochemiluminecence is light generated when a low voltage is applied to an electrode, thereby triggering a cyclic oxidation and reduction reaction of ruthenium metal ion bound in a chelate of tris-(bipyridine). This technology has broad applicability to detection of microbiological, biochemical and chemical analytes (Bruno et al., 1997, Rec. Res. Devel. In Micro. 1:25).
Electrochemiluminescent labels for DNA have been developed (Kenten et al., 1991, Clin. Chem. 37:1626; Kenten et al., 1992, Clin. Chem. 38:873; Origen(copyright) label, Origen(copyright) Phosphoramidite and TAG Phosphoramidite available from IGEN Inc., Rockville, Md.), and used for detection of polymerase chain reaction products (Schutzbank et al., 1995, J. Clin. Microbiol., 33:2023). These assays have been shown to be rapid and effective for the detection of amplified PCR products.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides a helicase assay that is rapid, sensitive and suitable for high throughput screening for potential helicase inhibitors. Helicase inhibitors represent a class of pharmacological agents useful for the treatment of disease.
Therefore, in a first embodiment, the present invention provides an electrochemiluminescent assay method for detecting helicase activity in a sample, the method comprising
(a) combining the sample with a first nucleic acid or nucleic acid molecule comprising an electrochemiluminescent label and hybridized to a complementary second nucleic acid or nucleic acid molecule;
(b) incubating the sample and hybridized nucleic acids or nucleic acid molecules from step (a) under conditions where helicase present in the sample can unhybridize the first and second nucleic acids or nucleic acid molecules;
(c) capturing unhybridized first nucleic acid or nucleic acid molecule; and
(d) measuring electrochemiluminescence from the captured first nucleic acid or nucleic acid molecule.
The helicase may be a human or pathogenic helicase. Preferably, the helicase is a fungal, viral, bacterial or parasitic helicase.
Any method for capturing unhybridized first nucleic acid or nucleic acid molecule may be employed. In a preferred series of steps, between steps (b) and (c) above, the unhybridized nucleic acids or nucleic acid molecules are incubated with a third nucleic acid or nucleic acid molecule complementary to the first nucleic acid or nucleic acid molecule and comprising a capturing ligand, under conditions where the first and third nucleic acids or nucleic acid molecules can hybridize. Thereafter, the hybridized first and third nucleic acids or nucleic acid molecules may be captured with a capturing receptor which binds the capturing ligand. The capturing receptor may be on the surface of magnetic beads. In a preferred embodiment, the capturing ligand is biotin and the capturing receptor is avidin. The preferred electrochemiluminescent label is a ruthenium chelate.
In another embodiment, the present invention relates to a method for detecting in a sample an agent that modulates helicase activity by
(a) combining the sample, a helicase and a first nucleic acid or nucleic acid molecule comprising an electrochemiluminescent label and hybridized to a complementary second nucleic acid or nucleic acid molecule, thereby forming a mixture;
(b) incubating the mixture from step (a) under conditions where the helicase can unhybridize the first and second nucleic acids or nucleic acid molecules in the absence of the sample;
(c) capturing unhybridized first nucleic acid or nucleic acid molecule; and
(d) measuring electrochemiluminescence from the captured first nucleic acid or nucleic acid molecule.
In a preferred embodiment, the agent is an inhibitor of helicase activity. The helicase may be a human or pathogenic helicase. Preferably, the helicase is a fungal, viral, bacterial or parasitic helicase.
Any method for capturing unhybridized first nucleic acid or nucleic acid molecule may be employed. In a preferred series of steps, between steps (b) and (c) above, the unhybridized nucleic acids or nucleic acid molecules are incubated with a third nucleic acid or nucleic acid molecule complementary to the first nucleic acid or nucleic acid molecule and comprising a capturing ligand, under conditions where the first and third nucleic acids or nucleic acid molecules can hybridize. Thereafter, the hybridized first and third nucleic acids or nucleic acid molecules may be captured with a capturing receptor which binds the capturing ligand. The capturing receptor may be on the surface of magnetic beads. In a preferred embodiment, the capturing ligand is biotin and the capturing receptor is avidin. The preferred electrochemiluminescent label is a ruthenium chelate.
In still another embodiment, the invention provides kits for electrochemiluminescent detection of helicase activity. Such kits comprise
(a) a first nucleic acid or nucleic acid molecule comprising an electrochemiluminescent label;
(b) a second nucleic acid or nucleic acid molecule complementary to the first nucleic acid or nucleic acid molecule;
(c) a helicase and/or magnetic beads suitable for facilitating electrochemiluminescence detection.
In one aspect, the first nucleic acid or nucleic acid molecule is already hybridized to the second nucleic acid or nucleic acid molecule. The kits according to the invention may further comprise a third nucleic acid or nucleic acid molecule complementary to the first nucleic acid or nucleic acid molecule and comprising a capturing ligand. Under such circumstances, the kit will, in general, include a capturing receptor capable of binding the capturing ligand. In a preferred embodiment, the capturing receptor is on the surface of magnetic beads. In a most preferred embodiment, the capturing ligand is biotin and the capturing receptor is avidin.